Para-hydroxycinnamic acid (pHCA) and cinnamic acid (CA) are multifunctional aromatic compounds that have potential utility in a wide variety of industrial applications. For example, these aromatic compounds have application as monomers for the production of liquid crystalline polymers, and in the production of resins, elastomers, coatings, adhesives, automotive finishes and inks.
Chemical synthetic methods for producing these aromatic compounds are known. However, these chemical methods are expensive due to the high cost of the starting materials and the extensive product purification required. Moreover, these methods generate large amounts of unwanted byproducts. Consequently, biological production methods for these aromatic compounds have been developed. For example, Gatenby et al. in U.S. Pat. No. 6,368,837 describe several methods for producing pHCA from glucose using bioengineered microorganisms. Additionally, Qi et al. in copending and commonly owner U.S. patent application Ser. No. 10/138,970 and U.S. Patent Application Publication No. 2003/007925 describe methods for producing CA and pHCA using recombinant microorganisms comprising at least one gene encoding a tyrosine ammonium lyase (TAL) activity and at least one gene encoding a phenylalanine hydroxylase (PAH) activity. However, a problem encountered with the biological production of these aromatic compounds is end-product inhibition, which limits product yield. Additionally, the fermentation is typically run at a pH that is not optimal for the tyrosine ammonium lyase activity, required to convert tyrosine to pHCA, or the phenylalanine ammonia lyase (PAL) activity, required to convert phenylalanine to CA. The pH optimum for these enzymes is in the alkaline pH range, typically about pH 8.5 (see for example Hodgins, J. Biol. Chem. 246:2977-2985 (1971)).
Evans et al. (Enzyme and Microbial Technology 9:417-421 (1987); Appl. Microbiol. Biotechnol. 25:399-405 (1987); and Journal of Industrial Microbiology. 2:53-58 (1987)) describe methods for the biotransformation of trans-cinnamic acid to phenylalanine using whole yeast cells, which have phenylalanine ammonia lyase activity, at a pH of 9.0 to 12.0. However, those disclosures do not describe the bioproduction of pHCA or CA in a two stage fermentation in which the pH is raised to alkaline values during the second stage of the fermentation.
One approach to mitigate end-product inhibition is to use two-phase extractive fermentation, in which the pHCA or CA produced by a recombinant production host is extracted into an immiscible organic phase during the fermentation so that it never reaches an inhibitory or critical concentration, as described by Ben Bassat et al. in copending and commonly owned U.S. patent application Ser. No. 10/824,237. The methods described in that disclosure resulted in improved yields for pHCA and CA. However, still higher yields are required for commercial applications.
Therefore, the need exists for a method for producing para-hydroxycinnamic acid and cinnamic acid in high yield for commercial applications.
Applicants have solved the stated problem by discovering methods for producing para-hydroxycinnamic acid and cinnamic acid in high yield using two-stage fermentation, wherein the pH is increased to alkaline values during the second stage of the fermentation.